Insulin lowers blood glucose by stimulating glucose uptake and storage in various target tissues, the most important being liver, skeletal muscle, and fat. The failure to respond appropriately to insulin results in a rise in blood sugar. Type II diabetes mellitus develops if beta cells become unable to release enough insulin to compensate for the insulin resistance. Determining the processes involved in the normal response to insulin will be essential for understanding insulin resistance, and the overall objective of this project is to define mechanisms involved in the actions of insulin in skeletal muscle and fat cells. Initial aims are to investigate the stimulation of glycogen synthesis by insulin. This effect is of particular importance in the control of blood glucose levels, as most of the glucose taken up following a meal is deposited as glycogen in skeletal muscle. Experiments in Aim 1 are proposed to investigate both the mechanism through which insulin activates glycogen synthase (GS), the enzyme that synthesizes glycogen from uridine diphosphoglucose (UDPG), and the importance of increasing GS activity in the stimulation of glycogen synthesis. Treating rats with insulin results in a marked decrease in muscle UDPG, implying that the activity of UDPG pyrophosphorylase (PPL) may limit the rate of glycogen synthesis. Objectives of Aim 2 are to determine whether UDPG PPL is subject to hormonal and/or metabolic control, and to investigate the potential limiting role of UDPG PPL in glycogen synthesis in rodent and human muscles. In the last two Aims we will investigate new targets of insulin action. We have recently discovered an adipocyte protein, designated betaip140, which is phosphorylated in response to insulin and coimmunoprecipitates with the beta isoform of protein kinase B (PKB). By purifying betaip140 and sequencing peptides by tandem mass spectrometry, we have shown that betaip140 is the product of the Kiaa0188 gene, recently identified by genetic fine mapping as a candidate gene for the fld mouse phenotype. Mice homozygous for the fld gene exhibit insulin resistance, glucose intolerance, and markedly diminished adipose tissue mass. Aim 3 is to investigate the potential interactions between betaip140 and PKBbeta, to define the mechanisms controlling betaip140 phosphorylation, and to determine the role of betaip140 in insulin action. Many other proteins that are phosphorylated in response to insulin can be detected, but have not been identified. This represents a serious gap in our understanding of insulin action, since at least some of the proteins are likely to represent downstream targets that are involved in the important metabolic responses to insulin. The objective of Aim 4 is to identify these new targets.